Regulation of Lhb and Egr1 gene expression by GNRH pulses in rat pituitaries is both c-Jun N-terminal kinase (JNK)- and extracellular signal-regulated kinase (ERK)-dependent - PubMed (original) (raw)
Regulation of Lhb and Egr1 gene expression by GNRH pulses in rat pituitaries is both c-Jun N-terminal kinase (JNK)- and extracellular signal-regulated kinase (ERK)-dependent
Laura L Burger et al. Biol Reprod. 2009 Dec.
Abstract
Pulsatile GNRH regulates the gonadotropin subunit genes in a differential manner, with faster frequencies favoring Lhb gene expression and slower frequencies favoring Fshb. Early growth response 1 (EGR1) is critical for Lhb gene transcription. We examined GNRH regulation of EGR1 and its two corepressors, Ngfi-A-binding proteins 1 and 2 (NAB1 and NAB2), both in vivo and in cultured rat pituitary cells. In rats, fast GNRH pulses (every 30 min) stably induced Egr1 primary transcript (PT) and mRNA 2-fold (P < 0.05) for 1-24 h. In contrast, slow GNRH pulses (every 240 min) increased Egr1 PT at 24 h (6-fold; P < 0.05) but increased Egr1 mRNA 4- to 5-fold between 4 and 24 h. Both GNRH pulse frequencies increased EGR1 protein 3- to 4-fold. In cultured rat pituitary cells, GNRH pulses (every 60 min) increased Egr1 (PT, 2.5- to 3-fold; mRNA, 1.5- to 2-fold; P < 0.05). GNRH pulses had little effect on Nab1/2 PT/mRNAs either in vivo or in vitro. We also examined specific intracellular signaling cascades activated by GNRH. Inhibitors of mitogen-activated protein kinase 8/9 (MAPK8/9 [also known as JNK]; SP600125) and MAP Kinase Kinase 1 (MAP2K1 [also known as MEK1]; PD98059) either blunted or totally suppressed the GNRH induction of Lhb PT and Egr1 PT/mRNA, whereas the MAPK14 (also known as p38) inhibitor SB203580 did not. In summary, pulsatile GNRH stimulates Egr1 gene expression and protein in vivo but not in a frequency-dependent manner. Additionally, GNRH-induced Egr1 gene expression is mediated by MAPK8/9 and MAPK1/3, and both are critical for Lhb gene transcription.
Figures
FIG. 1.
The effect of GNRH pulse frequency in vivo on Egr1, Nab1, and Nab2 PTs and mRNAs. Rats received 25-ng GNRH pulses either every 30 min or 240 min for 1–24 h (vehicle pulses to controls; n = 4–6 per group). Pituitaries were collected 5 min after the final pulse, and RNA was extracted. Primary transcripts or steady-state mRNAs were measured by quantitative real-time PCR. Data are expressed as percent 0-h controls ± SEM. Data were analyzed by two-way ANOVA with frequency and time as main effects. Basal levels (femtograms of plasmid/nanograms of RNA) for Egr1 PT and mRNA are 0.35 ± 0.06 and 34.0 ± 1.4; Nab1 PT and mRNA are 0.07 ± 0.01 and 0.8 ± 0.1; and Nab2 PT and mRNA are 0.70 ± 0.07 and 14.8 ± 1.01. *Significant difference (P < 0.05) vs. controls (0 h). **Significant difference between GNRH pulse-frequency paradigms at 24 h.
FIG. 2.
The effects of GNRH pulse frequency on EGR1 protein in vivo. Top: Representative Western blots of pituitary protein from rats pulsed with GNRH either every 30 min or 240 min for 8 h (n = 4–5 per group). Blots were immunostained for EGR1 and GAPDH. Protein amounts are 20 μg per lane. Bottom: Changes in EGR1 densitometry normalized to GAPDH for protein loading and expressed as percent 0-h (±SEM) controls. Points with different letters are significantly different (P < 0.05).
FIG. 3.
The effects of GNRH on Lhb and Fshb PTs, and Egr1, Nab1, and Nab2 PTs and mRNAs in cultured rat pituitary cells. Rat pituitary cells received two prepulses of GNRH (1 nM; media pulses to controls; 60-min interval). One hour later, cells were treated with 1 nM GNRH for 5–120 min (media to 0-h time point). Data are presented as percent (±SEM) of 0-h control (n = 4–5 per group). Groups marked with different letters are statistically different (P < 0.05). Basal levels (femtograms of plasmid/nanograms of RNA) for Lhb PT and Fshb PT are 0.59 ± 0.02 and 57.6 ± 2.9; Egr1 PT and mRNA are 4.7 ± 0.9 and 230.2 ± 22.6; Nab1 PT and mRNA are 0.15 ± 0.01 and 2.4 ± 0.2; and Nab2 PT and mRNA are 0.93 ± 0.06 and 24.2 ± 2.6.
FIG. 4.
The regulation of Egr1, Nab1, and Nab2 transcripts and EGR1 protein in αT3 cells. A) Gonadotroph-derived αT3 cells were treated with 10 nM GNRH for 10–120 min. After completing the study, cells were recovered, and PT/mRNA levels were determined. Data are presented as percent (±SEM) control (n = 6 per group). Groups marked with different letters are statistically different (P < 0.05). Basal levels (femtograms of plasmid/nanograms of RNA) for Egr1 PT and mRNA are 0.06 ± 0.01 and 3.4 ± 0.3; Nab1 PT and mRNA are 0.021 ± 0.002 and 7.4 ± 0.7; and Nab2 PT and mRNA are 0.004 ± 0.001 and 1.2 ± 0.1. B) Representative Western blots of protein from gonadotroph-derived αT3 cells treated as in A (n = 3 of 6 per group). Blots were immunostained for EGR1 and GAPDH. Protein amounts are 10% of culture lysate. C) Changes in EGR1 densitometry normalized to GAPDH for protein loading and expressed as percent 0-h (±SEM) controls. Points with different letters are significantly different (P < 0.05).
FIG. 5.
The effects of MAPK8/9 and MAPK14 blockade on Egr1, Nab1, and Nab2 PTs and mRNAs. Cultured rat pituitary cells received pulses of GNRH (200 pM; media pulses to controls; 60-min interval) in the presence of the MAPK8/9 blocker (SP600125; 20 μM), MAPK14 blocker (SB203580; 20 μM), or vehicle for 24 h. After completing the study, pituitary cells were recovered, and Egr1, Nab1, and Nab2 PTs and mRNAs were measured (n = 6 per group). Data are presented as percent (±SEM) control. Groups with different letters are statistically different (P < 0.05). Basal levels (femtograms of plasmid/nanograms of RNA) for Egr1 PT and mRNA are 2.7 ± 0.2 and 82.0 ± 5.8; Nab1 PT and mRNA are 0.09 ± 0.01 and 1.1 ± 0.1; and Nab2 PT and mRNA are 0.8 ± 0.1 and 29.0 ± 1.2.
FIG. 6.
The effects of MAPK8/9 and MAPK1/3 blockade on Egr1, Nab1, and Nab2 PTs and mRNAs. Rat pituitary cells were pretreated with: MAPK8/9 blocker (SP600125; 20 μM), MAP2K1 blocker (PD98059; 50 μM), or vehicle for 1 h. Then, cells received two prepulses of GNRH (1 nM; media pulses to controls; 60-min interval). One hour later, cells were treated with 1 nM GNRH for 10 min (media to controls). After completing the study, pituitary cells were recovered, and Lhb PT, Fshb PT, and Egr1, Nab1, and Nab2 PTs and mRNAs were measured (n = 4 per group). Data are presented as percent (±SEM) control. Groups marked with different letters are statistically different (P < 0.05). Basal levels (femtograms of plasmid/nanograms of RNA) for Lhb and Fshb PTs are 0.25 ± 0.01 and 63.2 ± 7.0; Egr1 PT and mRNA are 3.3 ± 0.5 and 153.1 ± 11.2; Nab1 PT and mRNA are 0.13 ± 0.02 and 3.5 ± 0.1; and Nab2 PT and mRNA are 0.60 ± 0.14 and 25.4 ± 1.2.
FIG. 7.
The effects of MAPK8/9 and MAPK1/3 blockade on EGR1, phosphorylated cJUN, and phosphorylated MAPK1/3. Top: Representative Western blots of protein from rat pituitary cells that were treated as in Figure 6 (n = 4 per group). Blots were immunostained for EGR1, phosphorylated cJUN, phosphorylated MAPK1/3, and GAPDH. Untreated αT3 protein lysates or αT3 cells plus 50 nM GNRH for 45 min were included as negative and positive controls, respectively. Protein amounts are 10% of culture lysate. Bottom: Changes in EGR1, phosphorylated cJUN, and phosphorylated MAPK1/3 densitometry normalized to GAPDH for protein loading and expressed as percent (±SEM) control. Points with different letters are significantly different (P < 0.05).
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